Chapter 3 │ Page 75 was added once after each splitting. Established HNSCC-PDOs HNSCC_001T and HNSCC_007T were used for experimental evaluation in this study (Table 3). Table 2. Full HNSCC-PDO medium. Basic medium consisted of Advanced DMEM (F12 12634, Life Technologies) with pen/strep (1%, 15140, Life Technologies), 1M Hepes (1%, 15630056, Life Technologies), and Glutamax (1%, 35050, Life Technologies). Basic medium was then further supplemented to obtain complete PDO medium. Cat nr Supplier Concentration B27 17504-044 Gibco 2% N-acetylcysteine A9165-5g Sigma 1.25 mM Nicotinamide N0636-100G Sigma 10 mM A83-01 2939 Tocris Bioscience 500 nM FGF-2 100-18B Peprotech 5 ng/mL FGF-10 100-26 Peprotech 10 ng/mL EGF AF-100-15 Peprotech 50 ng/ml Prostaglandin E2 2296 Tocris Bioscience 1 μM CHIR 99021 SML1046 Sigma 3 μM Forskolin 1099 Bio-Techne 1 μM Table 3. Patient and tumor characteristics of the included HNSCC-PDOs. Tumor Site Patient Details TNM Stage Pretreatment HNSCC_001T Primary Oral cavity (tongue base) Male, 60y T2 N1 M0 None HNSCC_007T Primary Hypopharynx Male, 61y T2 N1 M0 None 2.5.3. Organoid culturing and experimental handling Once successfully outgrown, organoids were passaged biweekly using TrypLE, dissociating them into single cells for subculturing in fresh cultrex domes. For experimental initiation, four-day-old organoids were gently harvested with cold organoid harvesting solution (Catalog # 3700-100-01, Bio-Techne), and thereafter plated out at 1500 organoids/well in in-house prepared 96-well plates pre-coated with cultrex. NTP and CDDP were administered as described in previous sections. Organoid kinetics were monitored every 4 hours up to 4 days post-treatment with live-cell imaging (Spark® Cyto), 0,05µM Cytotox Green reagent (4633, Essen BioScience) was used to follow up organoid death. The brightfield and green
RkJQdWJsaXNoZXIy MTk4NDMw